Visualizing Cytoskeletal Protein Reconstruction of Vulvar Cancer with Surface-Enhanced Raman Spectroscopy and Gold Nanoparticles

Metastasis of epithelial cancers often involves epithelial-mesenchymal transition (EMT), characterized by changes in cytoskeletal and adhesion protein expression. This includes the loss of the cell adhesion protein E-cadherin and the gain of the cytoskeletal protein vimentin. Our laboratory found that the epithelial vulvar cancer cell line A431 undergoes permanent loss of E-cadherin and gains vimentin expression when treated with a corticosteroid known as clobetasol (referred to as A431D cells). Clobetasol is commonly used to treat chronic vulvar rashes, making our findings significant when considering the repercussions of this treatment. Interestingly, the cells continued to express cytokeratin 8/18 in addition to vimentin. Raman spectroscopy has been used to monitor EMT in breast and oral cancer. We used 3-dimensional Surface-Enhanced Raman Scattering (SERS) imaging by utilizing colloidal gold nanoparticles. By tracking protein interactions and surface composition in cells before and after treatment with clobetasol, the distribution of the transition can be reasoned and visualized. Spectral assignments revealed the order of protein gains and losses, while the distinctness of the collected signals from the literature signals provided information about the folding, binding, and repelling forces between these cytoskeletal proteins. The three-dimensional Raman imaging of the cytoplasmic region of A431D cells exhibited unique features that combined the cytoarchitecture of A431 cells (parent cells expressing cytokeratins 8 and 18) and NIH 3T3 cells (used because they only express vimentin). This suggests that Raman imaging can be used to delineate cells that contain multiple types of intermediate filaments and serve as another method to identify cells undergoing EMT.